Although human hematopoietic stem cells (HSC) can be characterized by their activity in vitro and by their phenotypic expression of one or more antigens, they are most rigorously defined by their ability to reconstitute the hematopoietic system for the lifetime of the recipient under normal and (occasional) stress conditions. Because ethical and practical considerations prevent the testing of limiting numbers of human HSC in human recipients as has been successfully used for murine HSC, a number of surrogate assays using immunodeficient mice or preimmune fetal sheep have been developed to assess the in vivo engraftment and differentiation potentials of human HSC subsets. Of these, the fetal sheep model offers certain unique opportunities such as long-term observation, competitive engraftment, serial transfers, and provision of an early hematopoietic environment suitable for assessing the engraftment potential of putative HSC from embryonic sources, that are not easily achievable in the existing xenogeneic mouse assays. These attributes and the ability to achieve significant engraftment with relatively small numbers of human HSC from fetal, cord blood and adult sources have been used to advantage to assess the in vivo potential of human HSC subsets on a cell-per-cell basis. However, sheep require approved animal facilities not easily available in a majority of institutions. We have made the model available to a number of investigators often without cost. These collaborations have helped fine tune the model and establish its biological relevance as an assay for human HSC. The purpose of this application is to secure long-term funding for this large animal model by investigating 1) the effect of "humanizing" the model by pre- transplantation of human stromal/MSC cells on the engraftment and differentiation efficiency of human HSC; 2) human stem cell plasticity by studying the hematopoietic potential of human neuronal stem cells; and 3) whether mobilized human blood HSC differ from marrow HSC with regard to their engraftment potential on a cell-per-cell basis and whether any deficiency may be overcome by the use of large numbers of blood HSC. We also plan to determine whether the model can serve to detect HSC activity during human embryonic and early fetal development. We hope that these efforts will improve the availability and reliability of the sheep model, and develop new information with regard to the environmental control of HSC engraftment and/or differentiation in this model.